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Activation of P-TEFb by cAMP-PKA signaling in autosomal dominant polycystic kidney disease

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Science Advances  05 Jun 2019:
Vol. 5, no. 6, eaaw3593
DOI: 10.1126/sciadv.aaw3593
  • Fig. 1 cAMP-PKA signaling promotes P-TEFb release from P-TEFb/HEXIM1/7SK snRNP complex.

    (A) Co-immunoprecipitation (Co-IP) assays examining the interactions between FLAG-CDK9 and endogenous HEXIM1 in 293T cells with or without FSK treatment. (B) RNA-IP assays examining the associations between FLAG-CDK9 and 7SK snRNA in 293T cells with or without FSK treatment. (C) Co-IP assays examining the interactions between FLAG-HEXIM1 and endogenous P-TEFb in 293T cells treated with the indicated agents. (D) Co-IP assays examining the interactions between FLAG-HEXIM1 and endogenous P-TEFb in 293T cells transfected with vector or myc-PKA. (E) Co-IP assays examining the interactions between endogenous HEXIM1 and P-TEFb in 293T cells. (F) RNA-IP assays examining the associations between endogenous HEXIM1 (left) or P-TEFb (right) and 7SK snRNA in 293T cells as in (E). (G) Protein extracts from 293T cells transfected with vector or myc-PKA were separated by centrifugation through a 5 to 45% continuous glycerol gradient. Collected fractions were analyzed by Western blot analysis. Data represent as means ± SEM of three replicates in (B) and (F). IgG, immunoglobulin G.

  • Fig. 2 PKA-mediated Ser158phosphorylation of HEXIM1 disrupts the P-TEFb/HEXIM1/7SK snRNP complex.

    (A) PKA phosphorylates CDK9 and HEXIM1 but not CycT1. 293T cell lysates immunoprecipitated with the indicated antibodies were analyzed by Western blotting using a p-PKA substrate antibody. (B) Cross-species amino acid sequence alignment of HEXIM1. S* and T* denote phosphorylated serine and threonine, respectively. (C) In vitro PKA kinase assays containing GST-HEXIM1 (WT and S158A) as substrates. His/S-tag-PKA was detected by histidine (His) tag antibody. (D) Co-IP analysis of the interactions between the indicated FLAG-HEXIM1 and P-TEFb in 293T cells treated with or without FSK. (E) Western blot analysis of p-HEXIM1 and p-CREB in 293T cells treated with FSK. GAPDH, glyceraldehyde-phosphate dehydrogenase. (F) GST pulldown assays examining the interactions between CycT1 and GST-HEXIM1 (WT and S158A) with or without previous incubation with S-tag-PKA. (G) Co-IP assays examining the interactions between the indicated FLAG-HEXIM1 and other components of 7SK snRNP complex. (H) RNA-IP assays examining the associations between the indicated FLAG-HEXIM1 and 7SK snRNA. Data represent as means ± SEM of three replicates.

  • Fig. 3 P-TEFb kinase activity is increased in ADPKD kidneys.

    (A) Western blot analysis of p-HEXIM1 in kidneys from WT and Pkd1−/− mice. (B) Immunofluorescence analysis of p-HEXIM1 level in kidneys from WT and Pkd1−/− mice. (C) Western blot analysis of p-HEXIM1 in normal human kidneys and ADPKD kidneys. (D) Immunofluorescence analysis of p-HEXIM1 level in human kidneys. (E and F) Co-IP assays examining the interactions between P-TEFb and HEXIM1 in WT and Pkd1−/− mice kidneys. The tissue lysates were immunoprecipitated with CDK9 and CycT1 antibody in (E). Arrows indicated phosphorylated Pol II. (G) Kidney lysates immunoprecipitated by the anti–P-TEFb antibody were subjected to in vitro kinase assay, using GST-Pol II CTD as substrates. (H) Western blot analysis of kidney lysates from WT and Pkd1−/− mice. (I) Immunofluorescence analysis of Pol II CTD Ser2P in kidneys from WT and Pkd1−/− mice. (J) Western blot analysis of protein lysates from normal human kidneys and ADPKD kidneys. (K) Immunofluorescence analysis of Pol II Ser2P levels in human kidneys. Each gel line was obtained from an independent mouse (A and H) or human (C and J). “*” indicates nonspecific bands. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole.

  • Fig. 4 Disruption of the P-TEFb/HEXIM1/7SK snRNP complex accelerates cystogenesis in zebrafish pkd2 morphants.

    (A) Representative pictures of zebrafish embryos injected with the indicated MO. Notochord was indicated by “Nc.” Asterisks indicate dilated pronephric tubules. Pictures were taken at 2 days post fertilization (dpf). (B) Quantification of the incidences of pronephric cyst occurrence in zebrafish embryos from the indicated groups. (C) Western blot analysis of Pc2 and Hexim1 expression in zebrafish embryos from the indicated groups. The Pc2 density was normalized using the Gapdh level. (D) Representative pictures of zebrafish embryos injected with the indicated MOs and mRNAs. Pictures were taken at 2 dpf. (E) Quantification of the incidences of pronephric cyst occurrence in zebrafish embryos from the indicated groups. (F) Western blot analysis of Pc2, Hexim1, and exogenous EGFP-HEXIM1 levels in zebrafish embryos from the indicated groups. (G) Co-IP assays examining the interactions between exogenous EGFP-HEXIM1 (WT and S158E) and zebrafish CDK9. Scale bars, 200 μm (top) and 10 μm (bottom) in (A) and (D). Quantification data (n > 50) represent means ± SEM of three replicates in (B) and (E). *P < 0.05, **P < 0.01. Two-tailed unpaired Student’s t test was used for statistical analysis.

  • Fig. 5 FP treatment inhibits renal cyst growth in the early- and late-onset ADPKD mouse model.

    (A) Schematic view of experimental design in the early-onset ADPKD model. (B) Representative images of P29 kidneys from WT and Pkd1−/− mice treated with dimethyl sulfoxide (DMSO) or FP. (C) Ratios of kidney weight to body weight in the indicated groups of mice. (D) Hematoxylin and eosin (H&E) staining of kidney sections from Pkd1−/− mice treated with DMSO or FP. (E) Cystic index of H&E-stained kidney sections from Pkd1−/− mice treated with DMSO or FP. (F) Plasma BUN levels of P29 mice from the indicated groups. (G) Schematic view of the experimental design in the late-onset ADPKD model. (H) Representative magnetic resonance imaging (MRI) images of kidneys from the indicated groups. (I) Total kidney volumes (TKVs) were calculated from MRI images acquired at the indicated time points. (J) H&E staining of kidney sections from Pkd1−/− mice treated with DMSO or FP. (K) Cystic index of H&E-stained kidney sections as in (J). (L) Plasma BUN levels of P115 mice from the indicated groups. Scale bars, 2 mm (B, D, and J). Data represent as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. Two-tailed unpaired Student’s t test was used for statistical analysis. NS, not significant. (Photo credit: Zhiheng Liu, Tianjin Medical University).

  • Fig. 6 Inhibition of P-TEFb attenuates a pathological gene expression program of ADPKD by regulating Pol II pause release.

    (A to C) Volcano plots showing differentially expressed genes in kidneys from WT versus Pkd1−/− (A), WT DMSO versus WT FP (B), and Pkd1−/− DMSO versus Pkd1−/− FP (C) mice. (D) Heatmap of gene expression values of kidneys from the indicated groups. Rows show Z scores calculated for each group. (E and F) Boxplots of relative gene fragments per kilobase million (FPKM) values of kidneys from WT, WT FP, Pkd1−/−, and Pkd1−/− FP mice. (G) Gene tracks showing representative RNA-seq profiles of two genes (Col10a1 and Serpine1) in kidneys from the indicated groups. (H) Gene ontology enrichment analysis of FP-rescued genes. (I) Cumulative curve of Pol II pausing index (PI) for PKD-induced genes (left) and FP-rescued genes (right). (J) Gene tracks showing representative ChIP-Seq profiles of two genes (Pdk4 and Ctgf) in kidneys from the indicated groups. (K) Cumulative curve of relative pausing index for FP-rescued genes and nonrescued genes. (L) Working model of activation and function of P-TEFb in ADPKD. **P < 0.01, ***P < 0.001. One-way ANOVA with Tukey’s post hoc test was used for statistical analysis in (E) and (F). Two-tailed unpaired Student’s t test was used in (I) and (K).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/6/eaaw3593/DC1

    Supplementary Materials and Methods

    Fig. S1. PKA phosphorylates HEXIM1 at serine-158.

    Fig. S2. Phosphorylation of CDK9 at serine-347 by PKA does not affect P-TEFb/HEXIM1/7SK snRNP complex assembly.

    Fig. S3. The phosphorylation level of HEXIM1 is increased in cystic epithelial cells.

    Fig. S4. The interaction between P-TEFb and Hexim1 is conserved in zebrafish.

    Fig. S5. FP inhibits cystogenesis in the embryonic kidney model.

    Fig. S6. Inhibition of CDK9 attenuates cyst development in the MDCK cyst model.

    Fig. S7. HEXIM1 phosphorylation and P-TEFb/HEXIM1/7SK snRNP complex assembly in the MDCK cyst model.

    Fig. S8. Effect of FP on cystogensis in the early- and late-onset ADPKD mouse models.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. PKA phosphorylates HEXIM1 at serine-158.
    • Fig. S2. Phosphorylation of CDK9 at serine-347 by PKA does not affect P-TEFb/HEXIM1/7SK snRNP complex assembly.
    • Fig. S3. The phosphorylation level of HEXIM1 is increased in cystic epithelial cells.
    • Fig. S4. The interaction between P-TEFb and Hexim1 is conserved in zebrafish.
    • Fig. S5. FP inhibits cystogenesis in the embryonic kidney model.
    • Fig. S6. Inhibition of CDK9 attenuates cyst development in the MDCK cyst model.
    • Fig. S7. HEXIM1 phosphorylation and P-TEFb/HEXIM1/7SK snRNP complex assembly in the MDCK cyst model.
    • Fig. S8. Effect of FP on cystogensis in the early- and late-onset ADPKD mouse models.

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